Microglial Proteins Research Articles

SIRT1 activation by Ligustrazine ameliorates migraine via the paracrine interaction of microglia and neurons

BackgroundNeuroinflammation with associated oxidative stress aggravates the pathogenesis and progression of migraine. Ligustrazine (LGZ) is a key component from traditional edible-medicinal herb Ligusticum chuanxiong Hort., and has the effects of anti-platelet aggregation, expanding small arteries, improving microcirculation and promoting blood circulation and removing blood stasis in clinic. Hypothesis/PurposeThis study aims to investigate the pharmacological effect and mechanism of LGZ in migraine. Study Design/MethodsA mouse model of migraine was induced by nitroglycerin (NTG), and LPS/IFN-γ stimulated microglial cell model was conducted to investigate neuroinflammation, the paracrine interactions between microglia and neurons were determined by the co-culture system, and the effect of LGZ on stability of SIRT1 protein was measured by cellular thermal shift assay (CETSA). Whilst, the SIRT1 inhibitor EX527 was used alone or co-treatment with LGZ in vitro or in vivo. ResultsLGZ significantly attenuated migraine-like behaviors in NTG-induced mice, and ameliorated neuroinflammation and related oxidative damage in brain tissue, but co-treatment with SIRT1 inhibitor EX527 abolished the protective effects of LGZ. Mechanistically, LGZ mitigated neuroinflammation by upregulating SIRT1 expression and subsequently inhibiting the activation of NF-κB pathway in microglia. CETSA indicated that LGZ significantly maintained the stability of SIRT1 protein in microglia. While, in the co-culture system, culture medium from LPS/IFN-γ-treated microglia exacerbated neuronal damage and oxidative stress, which was suppressed by treating LPS/IFN-γ-induced microglia with LGZ, this effect might be related to the activation of Nrf2 signals in neurons. Notably, SIRT1 inhibitor EX527 abrogated the effects of LGZ both in vitro and in vivo. ConclusionConsequently, SIRT1 might be an important pharmacological target of LGZ, which attenuates migraine associated neuroinflammation and oxidative stress by interfering the crosstalk between microglia and neurons, thereby relieving migraine.

PPARβ/δ Agonist GW0742 Modulates Microglial and Astroglial Gene Expression in a Rat Model of Temporal Lobe Epilepsy.

The role of astroglial and microglial cells in the pathogenesis of epilepsy is currently under active investigation. It has been proposed that the activity of these cells may be regulated by the agonists of peroxisome proliferator-activated nuclear receptors (PPARs). This study investigated the effects of a seven-day treatment with the PPAR β/δ agonist GW0742 (Fitorine, 5 mg/kg/day) on the behavior and gene expression of the astroglial and microglial proteins involved in the regulation of epileptogenesis in the rat brain within a lithium-pilocarpine model of temporal lobe epilepsy (TLE). TLE resulted in decreased social and increased locomotor activity in the rats, increased expression of astro- and microglial activation marker genes (Gfap, Aif1), pro- and anti-inflammatory cytokine genes (Tnfa, Il1b, Il1rn), and altered expression of other microglial (Nlrp3, Arg1) and astroglial (Lcn2, S100a10) genes in the dorsal hippocampus and cerebral cortex. GW0742 attenuated, but did not completely block, some of these impairments. Specifically, the treatment affected Gfap gene expression in the dorsal hippocampus and Aif1 gene expression in the cortex. The GW0742 injections attenuated the TLE-specific enhancement of Nlrp3 and Il1rn gene expression in the cortex. These results suggest that GW0742 may affect the expression of some genes involved in the regulation of epileptogenesis.

Nrf2 Deficiency Exacerbates Parkinson's Disease by Aggravating NLRP3 Inflammasome Activation in MPTP-Induced Mouse Models and LPS-Induced BV2 Cells.

Parkinson's disease (PD) is a movement disorder characterized by the progressive loss of dopamine neurons. Microglia-mediated neuroinflammation drives disease progression and becomes a critical factor in neuronal degeneration. Recent studies have found that nuclear factor-erythroid 2-related-2 (Nrf2) expression levels are reduced during aging and neurodegenerative diseases, but its regulatory mechanism on microglia-induced neuroinflammation has not been fully elucidated. In vivo, we used the intraperitoneal injection of the neurotoxic drug neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to establish an animal model of PD and, at the same time, administered Nrf2 inhibitors ML385 and dimethyl fumarate to regulate Nrf2 protein levels. In vitro, we used si-RNA to knock out the Nrf2 gene to intervene in BV2 cells and used lipopolysaccharide (LPS) to stimulate and induce the cell model. The study found that inhibition of Nrf2 expression aggravated the motor defects of PD mice, accompanied by a significant loss of dopaminergic neurons in the substantia nigra and striatum of the brain. In addition, after inhibition of Nrf2, the malondialdehyde (MDA) level in the substantia nigra of the midbrain of mice increased, and the levels of superoxide dismutase (SOD) and heme oxygenase-1 (HO-1) decreased, accompanied by the proliferation of microglia and astrocytes. In addition, the activation of the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome, the assembly of apoptosis-associated speck-like protein containing a CARD (ASC) protein in microglia, and the release of downstream inflammatory factors caspase-1 and interleukin (IL)-1β, were aggravated. At the cellular level, it was found that knocking out the expression of Nrf2 would aggravate the activation of NLRP3 inflammasomes and the assembly of ASC in LPS-induced BV2 cells. Inhibited Nrf2 activity can reduce the downstream antioxidant enzyme HO-1 and antioxidant levels, induce NLRP3 inflammasome activation and ASC protein assembly in microglia, and ultimately aggravate PD inflammatory response and dopamine neuron degeneration.

Proteomic networks of gray and white matter reveal tissue-specific changes in human tauopathy.

To define tauopathy-associated changes in the human gray and white matter proteome. We applied tandem mass tagged labeling and mass spectrometry, consensus, and ratio weighted gene correlation network analysis (WGCNA) to gray and white matter sampled from postmortem human dorsolateral prefrontal cortex. The sampled tissues included control as well as Alzheimer's disease, corticobasal degeneration, progressive supranuclear palsy, frontotemporal degeneration with tau pathology, and chronic traumatic encephalopathy. Only eight proteins were unique to gray matter while six were unique to white matter. Comparison of the gray and white matter proteome revealed an enrichment of microglial proteins in the white matter. Consensus WGCNA sorted over 6700 protein isoforms into 46 consensus modules across the gray and white matter proteomic networks. Consensus network modules demonstrated unique and shared disease-associated microglial and endothelial protein changes. Ratio WGCNA sorted over 6500 protein ratios (white:gray) into 33 modules. Modules associated with mitochondrial proteins and processes demonstrated higher white:gray ratios in diseased tissues relative to control, driven by mitochondrial protein downregulation in gray and upregulation in white. The dataset is a valuable resource for understanding proteomic changes in human tauopathy gray and white matter. The identification of unique and shared disease-associated changes across gray and white matter emphasizes the utility of examining both tissue types. Future studies of microglial, endothelial, and mitochondrial changes in white matter may provide novel insights into tauopathy-associated changes in human brain.

Identification of distinct and shared biomarker panels in different manifestations of cerebral small vessel disease through proteomic profiling.

The pathophysiology underlying various manifestations of cerebral small vessel disease (cSVD) remains obscure. Using cerebrospinal fluid proximity extension assays and co-expression network analysis of 2,943 proteins, we found common and distinct proteomic signatures between white matter lesions (WML), microbleeds and infarcts measured in 856 living patients, and validated WML-associated proteins in three additional datasets. Proteins indicative of extracellular matrix dysregulation and vascular remodeling, including ELN, POSTN, CCN2 and MMP12 were elevated across all cSVD manifestations, with MMP12 emerging as an early cSVD indicator. cSVD-associated proteins formed a co-abundance network linked to metabolism and enriched in endothelial and arterial smooth muscle cells, showing elevated levels at early disease manifestations. Later disease stages involved changes in microglial proteins, associated with longitudinal WML progression, and changes in neuronal proteins mediating WML-associated cognitive decline. These findings provide an atlas of novel cSVD biomarkers and a promising roadmap for the next generation of cSVD therapeutics.

RGS10 mitigates high glucose-induced microglial inflammation via the reactive oxidative stress pathway and enhances synuclein clearance in microglia.

Microglia play a critical role in maintaining brain homeostasis but become dysregulated in neurodegenerative diseases. Regulator of G-protein Signaling 10 (RGS10), one of the most abundant homeostasis proteins in microglia, decreases with aging and functions as a negative regulator of microglia activation. RGS10-deficient mice exhibit impaired glucose tolerance, and high-fat diet induces insulin resistance in these mice. In this study, we investigated whether RGS10 modulates microglia activation in response to hyperglycemic conditions, complementing our previous findings of its role in inflammatory stimuli. In RGS10 knockdown (KD) BV2 cells, TNF production increased significantly in response to high glucose, particularly under proinflammatory conditions. Additionally, glucose uptake and GLUT1 mRNA levels were significantly elevated in RGS10 KD BV2 cells. These cells produced higher ROS and displayed reduced sensitivity to the antioxidant N-Acetyl Cysteine (NAC) when exposed to high glucose. Notably, both BV2 cells and primary microglia that lack RGS10 exhibited impaired uptake of alpha-synuclein aggregates. These findings suggest that RGS10 acts as a negative regulator of microglia activation not only in response to inflammation but also under hyperglycemic conditions.

Regulation of human microglial gene expression and function via RNAase-H active antisense oligonucleotides in vivo in Alzheimer’s disease

BackgroundMicroglia play important roles in maintaining brain homeostasis and neurodegeneration. The discovery of genetic variants in genes predominately or exclusively expressed in myeloid cells, such as Apolipoprotein E (APOE) and triggering receptor expressed on myeloid cells 2 (TREM2), as the strongest risk factors for Alzheimer’s disease (AD) highlights the importance of microglial biology in the brain. The sequence, structure and function of several microglial proteins are poorly conserved across species, which has hampered the development of strategies aiming to modulate the expression of specific microglial genes. One way to target APOE and TREM2 is to modulate their expression using antisense oligonucleotides (ASOs).MethodsIn this study, we identified, produced, and tested novel, selective and potent ASOs for human APOE and TREM2. We used a combination of in vitro iPSC-microglia models, as well as microglial xenotransplanted mice to provide proof of activity in human microglial in vivo.ResultsWe proved their efficacy in human iPSC microglia in vitro, as well as their pharmacological activity in vivo in a xenografted microglia model. We demonstrate ASOs targeting human microglia can modify their transcriptional profile and their response to amyloid-β plaques in vivo in a model of AD.ConclusionsThis study is the first proof-of-concept that human microglial can be modulated using ASOs in a dose-dependent manner to manipulate microglia phenotypes and response to neurodegeneration in vivo.

Emerging Microglial Therapies and Targets in Clinical Trial.

Modulation of microglia function for treatment of neurodegenerative and neuropsychiatric disorders is an emerging field of neuroscience drug development. This is largely attributed to human genetic association studies combined with biological evidence indicating that the innate immune system acts as a causal contributor superimposed on the reactive component of neuronal loss in neurological dysfunction. The identification of disease risk gene variants that encode immune-modulatory proteins in microglia provides tools to evaluate how microglia cellular function or dysfunction affect neuronal health. The development of clinical stage therapeutic compounds that modify myeloid cell function enables us to investigate how modulating microglia function could become a transformational approach to mitigate neurological disorders. Improving our ability to boost microglia-promoting homeostatic and reparative functions hopefully will translate into achieving a better outcome for patients affected by neurological diseases. In this chapter, we aim to provide an overview of the microglial emerging therapies and targets being studied in current clinical trials.

Pharmacological impact on the expression of microglial and astroglial proteins involved in the regulation of epileptogenesis as a possible new strategy for epilepsy therapy

Pharmacological impact on the expression of microglial and astroglial proteins involved in the regulation of epileptogenesis as a possible new strategy for epilepsy therapy

Selective vulnerability of the suprachiasmatic nucleus in progressive Alzheimer’s Disease: A human postmortem study using spatial in‐situ proteomics

AbstractBackgroundAlzheimer’s Disease (AD) patients commonly experience sleep/wake (S/W) disturbances, such as sleep fragmentation and excessive daytime sleepiness. The S/W cycle is controlled by a complex system regulated by a master regulator, the suprachiasmatic nucleus (SCN) that controls wake‐ and sleep‐promoting nuclei. Despite its importance in orchestrating sleep architecture, little is known about the molecular changes that occur in the SCN during the progression of AD and how these changes contribute to S/W disturbances.MethodWe used postmortem brain tissue of 10 controls without AD pathology and 32 subjects with progressive AD stages. Our approach included quantitative neuropathological assessments of AVP and VIP‐expressing neurons in SCN and its adjacent hypothalamic region, the supraoptic nucleus (SON). We also used an in‐situ cell‐specific spatial proteomics platform [GeoMx Digital Spatial Profiling (DSP)] to probe levels of different pathological tau species and proteins commonly dysregulated in AD.ResultPreliminary findings reveal a significant decrease in SCN volume (∼75%) (Fig.1) and a loss of ∼58% AVP neurons and ∼41% VIP neurons in Braak stage (Braak) VI compared to Braak 0. The DSP results indicate that both neurons in the SCN have twice the burden of p‐tau levels as the SON (Fig.2‐right), and neurofibrillary tanglesAdditionally, SCN shows an increase in proteins associated with glia and immune responses (Fig.2‐left). Furthermore, an increase in microglial proteins is observed in the SCN in Braak I (Fig.3‐left). In contrast, the SON displays a milder pattern of protein dysregulation in Braak I (Fig.3‐right), consistent with its lower tau expression observed in histology and DSP.ConclusionSCN is vulnerable to AD pathology even in Braak I, which is characterized by volume loss, increased immune response protein dysregulation, and later neuronal loss, as well as p‐tau pathology. This pattern supports the idea that the SCN plays a role in S/W disturbances observed from early AD stages. Interestingly, its neighboring nucleus, SON, which also has AVP neurons, remained spared, providing opportunities for studies investigating factors related to selective vulnerability. Ongoing studies are addressing changes in the SCN and SON in intermediate AD stages.

Microglial phenotype and proinflammatory gene induction in human prefrontal cortex in Alcohol Use Disorder overlap with Alzheimer’s Disease

AbstractBackgroundAlcohol Use Disorder (AUD) and heavy alcohol abuse are associated with dementia. However, studies focused on Alzheimer’s disease (AD) tend to exclude heavy alcohol drinking patients. We hypothesize microglial phenotype changes in AUD contribute to degeneration that parallels AD.MethodOrbital frontal cortex (OFC) from moderate drinking controls and heavy drinking AUD individuals paired by age were obtained from the New South Wales Tissue Resource Center in Sydney Australia. Tissue blocks were used for RTPCR gene mRNA and paraffin sections for immunohistochemistry assessment of microglial and proinflammatory genes and proteins, respectively. Fluoro‐Jade B, TUNEL stain and neuron marker NeuN were used to assess degeneration.ResultAUD OFC expressed increased levels of proinflammatory HMGB1, RAGE, and multiple Toll‐like receptors (TLR). In addition, AUD OFC increased expression of the TLR adaptor protein myeloid differentiation primary response 88 (MyD88), NFκB, and proinflammatory cytokines, chemokines, and their corresponding receptors. Iba1, a marker of monocytes and microglia, is increased in AUD, but not mRNA levels. Macrophage‐1 antigen, a complement receptor, is increased in AUD cortex as is CX3C chemokine receptor 1 (CX3CR1), the fractalkine receptor. OX42 staining (CD11b) in also increased. Microglial markers P2ry12, and CSF1R also show greater cellular staining in human AUD. The lysosomal scavenger receptor marker CD68 is also increased in AUD cortex. CX3CR1 and CD11b mRNA are increased in AUD cortex, but not CD68. Interestingly, TMEM119, a specific microglial marker, is decreased in AUD, both mRNA and protein staining in contrast to other markers increasing. Assessment of DNA oxidation using 8‐hydroxy‐2'‐deoxyguanosine (8‐OHdG) a biomarker of oxidative DNA/RNA damage 8‐OHdG+IR indicated AUD more than doubled cortical cells with this marker. Induction of HMGB1‐TLR‐MyD88‐NFκB proinflammatory signaling pathways, oxidative stress and microglial activation correlate with markers of neurodegeneration.ConclusionThe increased expression of TLR receptors, MyD88 and NFkB and increased proinflammatory cytokines are consistent with increased microglial activation and oxidative stress induced degeneration. Studies support alcohol activated microglia‐monocytes as driving loss of NeuN and other neurodegeneration markers. The reduced TMEM119 level with other markers increasing may indicate monocytes are replacing microglia in AUD cortex.

MCC950 alleviates seizure severity and angiogenesis by inhibiting NLRP3/ IL-1β signaling pathway-mediated pyroptosis in mouse model of epilepsy

Epilepsy is one of the most common serious chronic brain disorders, affecting up to 70 million people worldwide. Vascular disruption, including blood–brain barrier impairment and pathological angiogenesis, exacerbates its occurrence. However, its underlying mechanisms remain elusive. MCC950 is a specific small-molecule inhibitor that selectively blocks NLRP3 inflammatory vesicle activation across the blood–brain barrier, limits downstream IL-1β maturation and release, and exerts therapeutic effects across multiple diseases. In the present study, an epilepsy model was established by intraperitoneal administration of Kainic acid to adult male C57BL/6J wild-type mice. The results revealed that the epilepsy susceptibility of MCC950-treated mice was decreased, and neural damage following seizure episodes was reduced. In addition, immunofluorescence staining, RT-qPCR, and Western blot demonstrated that MCC950 inhibited the expression of the NLRP3 inflammasome and its related proteins in microglia, whereas microangiogenesis was found to be increased in the cerebral cortex and hippocampus of epileptic mice, and these effects could be reversed by MCC950. Furthermore, neurobehavioral impairment was observed in the epileptic mouse model, and MCC950 similarly alleviated the aforementioned pathological process. To the best of our knowledge, this is the first study to establish that pathological microangiogenesis is associated with NLRP3/IL-1β signaling pathway activation in a Kainic acid-induced epilepsy mouse model and that MCC950 administration attenuates the above-mentioned pathological changes and exerts neuroprotective effects. Therefore, MCC950 is a promising therapeutic agent for the treatment of epilepsy.

Lead exposure induced lipid metabolism disorders by regulating the lipophagy process in microglia.

Environmental lead (Pb) pollution is a worldwide public health problem and causes various diseases, especially neurodegenerative diseases. It is increasingly recognized that microglia-mediated neuroinflammation plays a crucial role in lead neurotoxicity, but the underlying mechanisms remain to be further explored. Recent studies indicated that cell metabolism, especially lipid metabolism, regulates many microglial functions, including cytokine secretion and phagocytosis. Whether lipid metabolism is involved in Pb-induced neuroinflammation is still unknown. In the current studies, we investigated the effects of Pb on microglial lipid metabolism by utilizing lipidomics. Histochemistry staining and oxygen consumption rate (OCR) were used to validate lipidomics results. Fenofibrate (FEN), a peroxisome proliferator-activated receptor-α (PPAR-α) agonist, was applied to investigate whether lipid metabolism regulation mitigated Pb's neuroinflammatory response. Microglial autophagic proteins were detected to investigate the role of lipophagy in Pb's effect on lipid metabolism. Our results showed that Pb exposure increased concentrations of various lipid metabolites and induced lipid metabolism disorders, especially in fatty acid metabolism. Pb caused lipid droplet (LD) accumulation and slightly enhanced fatty acid oxidation (FAO) in microglia. FEN pretreatment markedly inhibited Pb's effects on LDs and further mitigated Pb-induced inflammatory response by reducing pro-cytokines' expression and enhancing phagocytosis function. FEN intervention also inhibited Pb's neurotoxicity by improving cognition-related behaviors. Pb exposure induced an abnormal increase of autophagic proteins, but the FEN addition partially neutralized Pb's effects on autophagy. Our data indicate that the Pb-induced neuroinflammation is regulated by fatty acid metabolism via the lipophagy process. Therapies focusing on lipid metabolism regulation are powerful tactics in Pb toxicity prevention and treatment.

Long-term Effects of Cannabidiol and/or Fentanyl Exposure in Rats Submitted to Neonatal Pain

The current study aimed to evaluate anxiety behavior, hippocampal ionized calcium-binding adaptor molecule 1 (Iba1) and cannabinoid receptor 1 (CB1) gene expression, and nociceptive response in adulthood after a combination of fentanyl and cannabidiol (CBD) for nociceptive stimuli induced during the first week of life in rats. Complete Freund’s adjuvant-induced inflammatory nociceptive insult on postnatal day (PN) 1 and PN3. Both fentanyl and CBD were used alone or in combination from PN1 to PN7. Behavioral and nociceptive tests were performed at PN60 and PN62. The expression of the microglial calcium-binding proteins Iba1 and CB1 was detected in the hippocampus using reverse Quantitative polymerase chain reaction (qPCR) and immunohistochemistry. Our results suggest that the anxiety behavior response and immune activation in adult life depend on the CBD dose combined with fentanyl for the nociceptive stimuli induced during the first week of life. Treatment of neonatal nociceptive insult with CBD and opioids showed significant dose-dependent and male-female differences. The increased gene expression in the hippocampus of the analyzed cannabinoid gene supports this data. In addition, treatment with fentanyl led to an increase in CB1 protein expression. Moreover, the expression of Iba1 varied according to the administered dose of CBD and may or may not be associated with the opioid. A lower dose of CBD during the inflammatory period was associated with enhanced anxiety in adult life. PerspectiveThe treatment of nociceptive stimuli with CBD and opioids during the first week of life demonstrated significant sex differences in adult life on anxiety behavior and supraspinal pain sensitivity.

Effects of HMGB1/RAGE/cathespin B inhibitors on alleviating hippocampal injury by regulating microglial pyroptosis and caspase activation in neonatal hypoxic-ischemic brain damage.

Microglia play a crucial role in regulating neuroinflammation in the pathogenesis of neonatal hypoxic-ischemic brain damage (HIBD). Pyroptosis, an inflammatory form of programmed cell death, has been implicated in HIBD; however, its underlying mechanism remains unclear. We previously demonstrated that high-mobility group box 1 protein (HMGB1) mediates neuroinflammation and microglial damage in HIBD. In this study, we aimed to investigate the association between HMGB1 and microglial pyroptosis and elucidate the mechanism involved in rats with HIBD (both sexes were included) and in BV2 microglia subjected to oxygen-glucose deprivation. Our results showed that HMGB1 inhibition by glycyrrhizin (20 mg/kg) reduced the expression of microglial pyroptosis-related proteins, including caspase-1, the N-terminus fragment of gasdermin D (N-GSDMD), and pyroptosis-related inflammatory factors, such as interleukin (IL) -1β and IL-18. Moreover, HMGB1 inhibition resulted in reduced neuronal damage in the hippocampus 72 h after HIBD and ultimately improved neurobehavior during adulthood, as evidenced by reduced escape latency and path length, as well as increased time and distance spent in the target quadrant during the Morris water maze test. These results revealed that HIBD-induced pyroptosis is mediated by HMGB1/receptor for advanced glycation end products (RAGE) signaling (inhibition by FPS-ZM1, 1 mg/kg) and the activation of cathespin B (cat B). Notably, cat B inhibition by CA074-Me (5 mg/kg) also reduced hippocampal neuronal damage by suppressing microglial pyroptosis, thereby ameliorating learning and memory impairments caused by HIBD. Lastly, we demonstrated that microglial pyroptosis may contribute to neuronal damage through the HMGB1/RAGE/cat B signaling pathway in vitro. In conclusion, these results suggest that HMGB1/RAGE/cat B inhibitors can alleviate hippocampal injury by regulating microglial pyroptosis and caspase activation in HIBD, thereby reducing the release of proinflammatory mediators that destroy hippocampal neurons and induce spatial memory impairments.

The mitochondrial protein TSPO in Alzheimer’s disease: relation to the severity of AD pathology and the neuroinflammatory environment

The 18kD translocator protein (TSPO) is used as a positron emission tomography (PET) target to quantify neuroinflammation in patients. In Alzheimer’s disease (AD), the cerebellum is the pseudo-reference region for comparison with the cerebral cortex due to the absence of AD pathology and lower levels of TSPO. However, using the cerebellum as a pseudo-reference region is debated, with other brain regions suggested as more suitable. This paper aimed to establish the neuroinflammatory differences between the temporal cortex and cerebellar cortex, including TSPO expression. Using 60 human post-mortem samples encompassing the spectrum of Braak stages (I–VI), immunostaining for pan-Aβ, hyperphosphorylated (p)Tau, TSPO and microglial proteins Iba1, HLA–DR and MSR-A was performed in the temporal cortex and cerebellum. In the cerebellum, Aβ but not pTau, increased over the course of the disease, in contrast to the temporal cortex, where both proteins were significantly increased. TSPO increased in the temporal cortex, more than twofold in the later stages of AD compared to the early stages, but not in the cerebellum. Conversely, Iba1 increased in the cerebellum, but not in the temporal cortex. TSPO was associated with pTau in the temporal cortex, suggesting that TSPO positive microglia may be reacting to pTau itself and/or neurodegeneration at later stages of AD. Furthermore, the neuroinflammatory microenvironment was examined, using MesoScale Discovery assays, and IL15 only was significantly increased in the temporal cortex. Together this data suggests that the cerebellum maintains a more homeostatic environment compared to the temporal cortex, with a consistent TSPO expression, supporting its use as a pseudo-reference region for quantification in TSPO PET scans.

Bace1 Deletion in the Adult Reverses Epileptiform Activity and Sleep-wake Disturbances in AD Mice.

Alzheimer's disease (AD) increases the risk for seizures and sleep disorders. We show here that germline deletion of β-site amyloid precursor protein (APP) cleaving enzyme-1 (BACE1) in neurons, but not in astrocytes, increased epileptiform activity. However, Bace1 deletion at adult ages did not alter the normal EEG waveform, indicating less concern for BACE1 inhibition in patients. Moreover, we showed that deletion of Bace1 in the adult was able to reverse epileptiform activity in 5xFAD mice. Intriguingly, treating 5xFAD and APPNL-G-F/NL-G-F (APP KI) mice of either sex with one BACE1 inhibitor Lanabecestat (AZD3293) dramatically increased epileptiform spiking, likely resulting from an off-target effect. We also monitored sleep-wake pathologies in these mice and showed increased wakefulness, decreased non-rapid eye movement sleep, and rapid eye movement sleep in both 5xFAD and APP KI mice; BACE1 inhibition in the adult 5xFAD mice reversed plaque load and sleep disturbances, but this was not seen in APP KI mice. Further studies with and without BACE1 inhibitor treatment showed different levels of plaque-associated microgliosis and activated microglial proteins in 5xFAD mice compared with APP KI mice. Together, BACE1 inhibition should be developed to avoid off-target effect for achieving benefits in reducing epileptic activity and sleep disturbance in Alzheimer's patients.SIGNIFICANCE STATEMENT BACE1 is widely recognized as a therapeutic target for treating Alzheimer's disease patients. However, BACE1 inhibitors failed in clinical trials because of inability to show cognitive improvement in patients. Here we show that BACE1 inhibition actually reduces sleep disturbances and epileptic seizures; both are seen in AD patients. We further showed that one of clinically tested BACE1 inhibitors does have off-target effects, and development of safer BACE1 inhibitors will be beneficial to AD patients. Results from this study will provide useful guidance for additional drug development.

IRAK-M Ablation Promotes Status Epilepticus-Induced Neuroinflammation via Activating M1 Microglia and Impairing Excitatory Synaptic Function.

Epilepsy is one of the most common neurological disorders. The pro-epileptic and antiepileptic roles of microglia have recently garnered significant attention. Interleukin-1 receptor-associated kinase (IRAK)-M, an important kinase in the innate immune response, is mainly expressed in microglia and acts as a negative regulator of the TLR4 signaling pathway that mediates the anti-inflammatory effect. However, whether IRAK-M exerts a protective role in epileptogenesis as well as the molecular and cellular mechanisms underlying these processes are yet to be elucidated. An epilepsy mouse model induced by pilocarpine was used in this study. Real-time quantitative polymerase chain reaction and western blot analysis were used to analyze mRNA and protein expression levels, respectively. Whole-cell voltage-clamp recordings were employed to evaluate the glutamatergic synaptic transmission in hippocampal neurons. Immunofluorescence was utilized to show the glial cell activation and neuronal loss. Furthermore, the proportion of microglia was analyzed using flow cytometry. Seizure dynamics influenced the expression of IRAK-M. Its knockout dramatically exacerbated the seizures and the pathology in epilepsy and increased the N-methyl-d-aspartate receptor (NMDAR) expression, thereby enhancing glutamatergic synaptic transmission in hippocampal CA1 pyramidal neurons in mice. Furthermore, IRAK-M deficiency augmented hippocampal neuronal loss via a possible mechanism of NMDAR-mediated excitotoxicity. IRAK-M deletion promotes microglia toward the M1 phenotype, which resulted in high levels of proinflammatory cytokines and was accompanied by a visible increase in the expressions of key microglial polarization-related proteins, including p-STAT1, TRAF6, and SOCS1. The findings demonstrate that IRAK-M dysfunction contributes to the progression of epilepsy by increasing M1 microglial polarization and glutamatergic synaptic transmission. This is possibly related to NMDARs, particularly Grin2A and Grin2B, which suggests that IRAK-M could serve as a novel therapeutic target for the direct alleviation of epilepsy.

Huoluo Xiaoling Pellet promotes microglia M2 polarization through increasing MCPIP1 expression for ischemia stroke alleviation

Huoluo Xiaoling Pellet (HXP), a Chinese patent medicine, is commonly administered for the treatment of treat ischemic strokes. MCPIP1, an inducible suppressor of the inflammatory response, is a regulator of microglial M2 polarization. This study aimed to explore whether HXP can promote microglial M2 polarization by upregulating MCPIP1 expression, consequently mitigating cerebral ischemic injury. Our study involved 85 Sprague-Dawley rats (weighing 250–280 g). We established middle cerebral artery occlusion (MCAO) and oxygen-glucose deprivation-reoxygenation (OGD/R) models with MCPIP1 knockdown to assess the effects of HXP on ischemic strokes. Our findings show that HXP reduced brain water content, improved neurological function, and inhibited the expression of inflammatory factors in the brain tissues of MCAO rats. The neuroprotective effects of HXP on cerebral ischemic injuries were compromised by MCPIP1 knockdown. Immunofluorescence results indicated that the expression of microglia marker Iba1 and M2 phenotypic marker CD206 was upregulated in MCAO rats and OGD/R-treated microglia. Administration of HXP significantly reduced Iba1 expression and facilitated CD206 expression, an effect that was counteracted by sh-MCPIP1 transfection. Western blotting revealed that HXP treatment augmented the expression of MCPIP1, microglial M2 marker proteins (CD206 and Arg1), and PPARγ, while reducing the expression of microglial M1 marker proteins (CD16 and iNOS) in MCAO rats and OGD/R-induced microglia. MCPIP1 knockdown suppressed HXP-mediated upregulation of MCPIP1, CD206, Arg1, and PPARγ, as well as the downregulation of CD16 and iNOS. Our findings suggest that HXP primarily ameliorates ischemic stroke through the upregulation of MCPIP1, which in turn induces microglial M2 polarization.

Cellular Proteomic Profiling Using Proximity Labeling by TurboID-NES in Microglial and Neuronal Cell Lines

Different brain cell types play distinct roles in brain development and disease. Molecular characterization of cell-specific mechanisms using cell type-specific approaches at the protein (proteomic) level can provide biological and therapeutic insights. To overcome the barriers of conventional isolation-based methods for cell type-specific proteomics, invivo proteomic labeling with proximity-dependent biotinylation of cytosolic proteins using biotin ligase TurboID, coupled with mass spectrometry (MS) of labeled proteins, emerged as a powerful strategy for cell type-specific proteomics in the native state of cells without the need for cellular isolation. To complement invivo proximity labeling approaches, invitro studies are needed to ensure that cellular proteomes using the TurboID approach are representative of the whole-cell proteome and capture cellular responses to stimuli without disruption of cellular processes. To address this, we generated murine neuroblastoma (N2A) and microglial (BV2) lines stably expressing cytosolic TurboID to biotinylate the cellular proteome for downstream purification and analysis using MS. TurboID-mediated biotinylation captured 59% of BV2 and 65% of N2A proteomes under homeostatic conditions. TurboID labeled endolysosome, translation, vesicle, and signaling proteins in BV2 microglia and synaptic, neuron projection, and microtubule proteins in N2A neurons. TurboID expression and biotinylation minimally impacted homeostatic cellular proteomes of BV2 and N2Acells and did not affect lipopolysaccharide-mediated cytokine production or resting cellular respiration in BV2 cells. MS analysis of the microglial biotin-labeled proteins captured the impact of lipopolysaccharide treatment (>500 differentially abundant proteins) including increased canonical proinflammatory proteins (Il1a, Irg1, and Oasl1) and decreased anti-inflammatory proteins (Arg1 and Mgl2).